Kurian Joseph R, Chin Nathaniel A, Longlais Brett J, Hayes Kristie L, Trepanier Lauren A
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706-1102, USA.
Chem Res Toxicol. 2006 Oct;19(10):1366-73. doi: 10.1021/tx060106t.
Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.
杂环和芳香胺类致癌物被认为是通过形成DNA加合物导致肿瘤起始，而生物活化生成芳基羟胺代谢产物对于与DNA发生反应是必需的。致癌性芳基羟胺代谢产物在人体内通过一种微粒体、依赖NADH、对氧不敏感的还原途径清除，这可能是个体对芳香胺类致癌物反应存在差异的一个来源。本研究的目的是确定人类肝脏中这种还原途径的特性。基于我们对治疗药物结构相似的芳基羟胺代谢产物的研究结果，我们推测芳基羟胺类致癌物的还原性解毒是由NADH细胞色素b5还原酶（b5R）和细胞色素b5（细胞色素b5）催化的。我们发现，芳香胺4-氨基联苯（4-ABP；存在于香烟烟雾中）和杂环胺2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP；存在于烤肉中）的致癌性羟胺的还原确实是由仅包含人b5R和细胞色素b5的纯化系统催化的。与人类肝脏微粒体（HLM）相比，纯化系统中的比活性高56-346倍，两个系统中的米氏常数（K(m)值）相似。在纯化系统中产生最高活性的b5R和细胞色素b5的化学计量比也与天然HLM中的相似（约为1:8至1:10）。针对b5R或细胞色素b5的多克隆抗血清分别显著抑制N-羟基-4-氨基联苯（NHOH-4-ABP）还原95%和89%，并且个体HLM中免疫反应性细胞色素b5蛋白含量与NHOH-4-ABP和N-羟基-PhIP（NHOH-PhIP）的个体还原显著相关。最后，将HLM滴定到纯化的b5R/细胞色素b5系统中并没有提高还原活性的效率。我们得出结论，b5R和细胞色素b5共同仅能够还原芳基羟胺类致癌物，并且我们进一步推测该途径可能是4-ABP或PhIP暴露后癌症易感性个体差异的一个来源。
